Project Summary Abuse of psychostimulants, including amphetamines (AMPHs), is a major public health problem with profound psychiatric, medical and psychosocial complications. Genetic factors contribute substantially to an individual's susceptibility to developing addiction; however, the search for risk alleles has yielded limited success. The initial sensitivity to psychostimulants varies significantly, and has been associated with continued use and abuse. This trait can be studied in animal models, which have emerged as powerful tools to investigate the behavioral response to drugs in a controlled and systematic manner. The combination of approaches we propose in this application will allow us to harness the power of Drosophila genetics to uncover novel genes and gene variants that confer sensitivity to AMPH. With its rapid life cycle and accessibility to genetic, cellular and molecular analyses, the fly has enabled behavioral studies in areas that are far more difficult to investigate in vertebrate animal models. The preliminary data presented in this application show that that flies respond to AMPH by increasing their locomotor activity and decreasing their sleep. Genetic mutations that disrupt dopamine (DA) synthesis or dopamine transporter gene (DAT) function inhibit these behavioral responses, demonstrating that we have developed a robust behavioral tool to associate genetic variations with phenotypic changes. We have developed a strategy combining this behavioral analysis with next-generation (Next-Gen) sequencing technology and systems genetics approaches to investigate the genetic architecture of AMPH sensitivity and identify new gene variants that influence this trait. This integrated approach is made possible by our active collaboration with Dr. David Goldman and Dr. Colin Hodgkinson at the Laboratory of Neurogenetics at NIAAA, experts in state-of-the-art Next-Gen technologies, genetic linkage studies and functional genomics approaches to the study of behavioral traits. We propose the following specific aims: 1) To identify gene variants that underlie wide variation in AMPH sensitivity within and between substrains of the wild- type, non-isogenic Drosophila strain Canton S (CS). We will (a) use selective breeding, combined with genomic approaches such as whole genome sequencing and deficiency mapping, to identify gene variants that alter AMPH sensitivity in different CS substrains and b) use RNA-sequencing (RNA-seq) to profile gene expression changes associated with altered sensitivity to AMPH in the different substrains and 2) To screen a large population of inbred strains to identify genetic loci associated with altered sensitivity to AMPH. We will (a) screen the Drosophila Genetic Reference Panel, which consists of 203 genotyped inbred lines, for response to AMPH and (b) use systems genetics approaches, including quantitative trait locus (QTL) analysis and extreme QTL mapping, to associate phenotypic variation in sensitivity to AMPH with DNA polymorphisms.